In vivo analysis of astroctye-neuron dynamics in circuit formation, function, and maintenance

项目摘要 哺乳动物的大脑是由数十亿个神经元组成的，这些神经元通过专门的化学连接点进行交流。 叫做突触。单个神经元连接起来形成正确学习所需的功能电路 和记忆，神经回路的中断是患者经历的衰弱症状的基础 患有癫痫和精神分裂症等神经系统疾病。虽然适当的形成和 神经回路的维持对于高质量的人类生活是必不可少的，这一过程使给定的 神经元找到正确的突触对，以及这些突触如何随着时间的推移而保持和修改， 不太了解。我们的实验室和其他人最近的工作已经确定了星形胶质细胞，最丰富的中枢神经系统 神经胶质细胞类型，作为突触发育的主要调节器。星形胶质细胞都是促突触发生的（例如， 星形胶质细胞导致突触发生减少）以及抗突触发生（例如星形胶质细胞吞噬和修剪 突触）。星形胶质细胞在调节突触数量方面的这些重要功能表明星形胶质细胞可能 调节更广泛的电路形成，尽管这一假设尚未得到充分研究。 行为相关回路中星形胶质细胞-神经元动力学的表征尚未被 进行，可能是因为它需要在体内操纵一对定义的突触耦合神经元 和相关的星形胶质细胞。考虑到哺乳动物神经系统的巨大复杂性，这些类型 在哺乳动物身上做实验还不可行。令人兴奋的是，现在可以在 果蝇神经系统由于最近开发的工具，星形胶质细胞的操作， 弗里曼实验室，并在Doe实验室识别控制幼虫运动的神经回路。作为一个共同指导的 作为Doe和Freeman实验室的博士后研究员，我将合并这些新工具， 可视化和遗传操纵单个中央突触的能力，我将与之结合 相关星形胶质细胞的靶向操作以确定星形胶质细胞在突触形成中的作用， 维护和功能。对于所有的研究，我将使用最近确定的标记定义的转基因品系， 突触对：E2和SA 1中间神经元之间的兴奋性胆碱能突触，以及抑制性， A31 k中间神经元与RP 2运动神经元之间的GABA能突触。星形胶质细胞将使用 抗Gat免疫荧光或UAS-myr：：Cerulean在alrm-GAL 4下的表达。 在我的第一个目标中，我将把星形胶质细胞消融实验与突变分析结合起来，以测试 在兴奋性和抑制性突触的发育（形成）中起作用的星形胶质细胞。在我的第二个目标，我 将使用光遗传学策略来测量兴奋性和抑制性突触的活性（功能）， 对星形胶质细胞功能变化的反应。最后，在我的第三个目标中，我将操纵神经元活动（通过 定义的突触前神经元的组成性激活或沉默），并测试神经元 活性影响星形胶质细胞的形态和功能。总之，这些实验将定义体内 星形胶质细胞在脑内兴奋性和抑制性突触的形成、功能和维持中的作用 行为相关的感觉运动回路

PROJECT SUMMARY The mammalian brain is formed by billions of neurons which communicate at specialized chemical junctions called synapses. Individual neurons connect to form functional circuits which are required for proper learning and memory, and disruption of neuronal circuitry underlies the debilitating symptoms experienced by patients suffering from neurological disorders such as epilepsy and schizophrenia. Although proper formation and maintenance of neuronal circuits is essential for a high quality of human life, the process by which a given neuron finds the correct synaptic pair, and how these synapses are maintained and modified over time is poorly understood. Recent work from our labs and others have identified astrocytes, the most abundant CNS glial cell type, as a major regulator of synaptic development. Astrocytes are both pro-synaptogenic (e.g. loss of astrocytes results in decreased synaptogenesis) as well as anti-synaptogenic (e.g. astrocytes engulf and prune synapses). These important functions of astrocytes in regulating synapse number suggest that astrocytes may regulate broader circuit formation, though this hypothesis has not been fully investigated. Characterization of astrocyte-neuron dynamics within a behaviorally-relevant circuit has not been performed, probably because it requires in vivo manipulation of a defined pair of synaptically-coupled neurons and the associated astrocytes. Given the enormous complexity of the mammalian nervous system, these types of experiments are not yet feasible in mammals. Excitingly, it is now possible to perform these studies in the Drosophila nervous system due to the recent development of tools for astrocyte manipulation from the Freeman lab, and identification of neural circuits governing larval locomotion in the Doe lab. As a co-mentored postdoctoral fellow within the Doe and Freeman laboratories, I will merge these new tools to have the unique ability to visualize and genetically manipulate individual central synapses, which I will couple with targeted manipulation of the associated astrocytes to define the role of astrocytes in synapse formation, maintenance, and function. For all studies, I will use recently identified transgenic lines that label defined synaptic pairs: the excitatory cholinergic synapses between E2 and SA1 interneurons, and the inhibitory, GABAergic synapses between A31k interneuron and RP2 motor neuron. Astrocytes will be visualized using anti-Gat immunofluorescence or expression of UAS-myr::Cerulean under alrm-GAL4. In my first aim, I will couple astrocyte ablation experiments with mutant analyses to test the necessity of functional astrocytes in the development (formation) of excitatory and inhibitory synapses. In my second aim, I will use an optogenetic strategy to measure the activity (function) of excitatory and inhibitory synapses in response to changes in astrocyte function. Finally, in my third aim, I will manipulate neuronal activity (through constitutive activation or silencing of defined pre-synaptic neurons) and test the hypothesis that neuronal activity influences both astrocyte morphology and function. In sum, these experiments will define the in vivo role of astrocytes in the formation, function, and maintenances of excitatory and inhibitory synapses within a behaviorally-relevant, sensorimotor circuit.